1. Field of the Invention
The present invention relates to structural analysis of nucleic acids, and more particularly to an apparatus and a method for detecting denaturation of nucleic acids.
2. Related Background Art
Much attention has been given to techniques for detecting and measuring denaturation of nucleic acids (DNA, RNA, and hybrids thereof) with accuracy which substantially resolves a difference in the denaturation conditions based on a single base (or single base-pair) substitution, deletion or addition.
For the detection of slight differences between homologous double-stranded nucleic acids, such as the above-mentioned single base alteration, there has been proposed a method called "denaturing gradient gel electrophoresis method" (S. G. Fischer and L. S. Lerman: Proc. Natl. Acid. Sci. USA, Vol.80, pp.1579-1583, March 1983). In this method, a double-stranded nucleic acid to be measured is first amplified using an ordinary technique such as polymerase chain reaction (hereinafter, referred to as "PCR method"). Then, the resultant double-stranded nucleic acid is charged into a gel carrier (or gel support), and the double-stranded nucleic acid is subjected to electrophoresis under a gradient of a denaturation condition (such as temperature or hydrogen ion concentration (pH)) which is spatially provided across the gel carrier. In this measurement, it is possible to simultaneously subject a control double-stranded nucleic acid functioning as a reference (or standard) together with the double-stranded nucleic acid to be measured so as to observe a difference in the denaturation condition between these two species of the double-stranded nucleic acids (i.e., the double-stranded nucleic acid to be measured and the reference double-stranded nucleic acid).
FIGS. 9A1-9A3 and 9B are schematic diagrams illustrating the above-mentioned conventional method for detecting the denaturation of a nucleic acid. For example, FIGS. 9A1-9A3 relate to a case wherein temperature is selected as a denaturation condition, and schematically shows a state wherein a nucleic acid is denatured on the basis of temperature increase. In general, a double-stranded nucleic acid is denatured when the ambient temperature is elevated to a predetermined value. The temperature at which a double-stranded nucleic acid is denatured (denaturation temperature) depends on the composition of bases constituting the double-stranded nucleic acid. A double-stranded nucleic acid comprising a specific single-stranded nucleic acid and another single-stranded nucleic acid binding thereto which is completely complementary to the former single-stranded nucleic acid, has a denaturation temperature higher than that of a double-stranded nucleic acid comprising the above-mentioned specific single-stranded nucleic acid and another single-stranded nucleic acid binding thereto which is substantially (and not completely) complementary to the former single-stranded nucleic acid.
Conventional apparatuses for detecting denaturation of a nucleic acid utilize such a phenomenon, and conduct gel electrophoresis while spatially providing a gradient of a denaturation condition in the gel carrier to be used for the electrophoresis.
FIG. 9B shows results of measurement relating to a case of a double-stranded nucleic acid sample comprising a specific stranded and another stranded which is substantially complementary to the specific stranded. As shown in FIG. 9B, when two denaturation points (i.e., points at which denaturation occurs) are present, one double-stranded nucleic acid showing a denaturation condition of a lower temperature is the double-stranded nucleic acid to be measured, and the other double-stranded nucleic acid showing a higher temperature denaturation is the reference double-stranded nucleic acid.
However, in the above-mentioned denaturation detection of a nucleic acid utilizing the conventional denaturing gradient gel electrophoresis method, it is necessary to subject the sample nucleic acid to electrophoresis in a gel, across which a gradient or change in denaturation condition (temperature or pH) is provided. Accordingly, such a conventional denaturation detection method is troublesome or tedious. More specifically, the conventional method consumes a long period of time corresponding to several tens of minutes to several hours, while such a period of time depends on the molecular weight of the sample nucleic acid.
In addition, the sensitivity of detection of the nucleic acid denaturation by the conventional denaturing gradient gel electrophoresis method depends on the stability of the system to be used for such a purpose, the accuracy in the control of the denaturation condition such as degree of gradient to be provided to the gel, or the accuracy in the control of the condition for electrophoresis. However, it is difficult to improve the accuracy in the control of these conditions as compared with those accomplished at the present stage. Accordingly, there has been posed a problem such that further improvement is difficult with respect to the sensitivity and accuracy (or precision) of the denaturation detection.